Isolated human drug-metabolizing proteins, nucleic acid molecules encoding human drug-metabolizing proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the drug-metabolizing enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the drug-metabolizing enzyme peptides, and methods of identifying modulators of the drug-metabolizing enzyme peptides.

FIELD OF THE INVENTION

The present invention is in the field of drug-metabolizing proteins thatare related to the UDP-glycosyltransferase drug-metabolizing enzymesubfamily, recombinant DNA molecules and protein production. The presentinvention specifically provides novel drug-metabolizing peptides andproteins and nucleic acid molecules encoding such protein molecules, foruse in the development of human therapeutics and human therapeuticdevelopment.

BACKGROUND OF THE INVENTION

Drug-Metabolizing Proteins

Induction of drug-metabolizing enzymes (“DMEs”) is a common biologicalresponse to xenobiotics, the mechanisms and consequences of which areimportant in academic, industrial, and regulatory areas of pharmacologyand toxicology.

For most drugs, drug-metabolizing enzymes determine how long and howmuch of a drug remains in the body. Thus, developers of drugs recognizethe importance of characterizing a drug candidate's interaction withthese enzymes. For example, polymorphisms of the drug-metabolizingenzyme CYP2D6, a member of the cytochrome p450 (“CYP”) superfamily,yield phenotypes of slow or ultra-rapid metabolizers of a wide spectrumof drugs including antidepressants, antipsychotics, beta-blockers, andantiarrhythmics. Such abnormal rates of drug metabolism can lead to drugineffectiveness or to systemic accumulation and toxicity.

For pharmaceutical scientists developing a candidate drug, it isimportant know as early as possible in the design phase which enzymesmetabolize the drug candidate and the speed with which they do it.Historically, the enzymes on a drug's metabolic pathway were determinedthrough metabolism studies in animals, but this approach has now beenlargely supplanted by the use of human tissues or cloneddrug-metabolizing enzymes to provide insights into the specific role ofindividual forms of these enzymes. Using these tools, the qualitativeand quantitative fate of a drug candidate can be predicted prior to itsfirst administration to humans. As a consequence, the selection andoptimization of desirable characteristics of metabolism are possibleearly in the development process, thus avoiding unanticipated toxicityproblems and associated costs subsequent to the drug's clinicalinvestigation. Moreover, the effect of one drug on another's dispositioncan be inferred.

Known drug-metabolizing enzymes include the cytochrome p450 (“CYP”)superfamily, N-acetyl transferases (“NAT”), UDP-glucuronosyltransferases (“UGT”), methyl transferases, alcohol dehydrogenase(“ADH”), aldehyde dehydrogenase (“ALDH”), dihydropyrimidinedehydrogenase (“DPD”), NADPH:quinone oxidoreductase (“NQO” or “DTdiaphorase”), catechol O-methyltransferase (“COMT”), glutathioneS-transferase (“GST”), histamine methyltransferase (“HMT”),sulfotransferases (“ST”), thiopurine methyltransferase (“TPMT”), andepoxide hydroxylase. Drug-metabolizing enzymes are generally classifiedinto two phases according to their metabolic function. Phase I enzymescatalyze modification of functional groups, and phase II enzymescatalyze conjugation with endogenous substituents. These classificationsshould not be construed as exclusive nor exhaustive, as other mechanismsof drug metabolism have been discovered. For example, the use of activetransport mechanisms been characterized as part of the process ofdetoxification.

Phase I reactions include catabolic processes such as deamination ofaminases, hydrolysis of esters and amides, conjugation reactions with,for example, glycine or sulfate, oxidation by the cytochrome p450oxidation/reduction enzyme system and degradation in the fatty acidpathway. Hydrolysis reactions occur mainly in the liver and plasma by avariety of non-specific hydrolases and esterases. Both deaminases andamidases, also localized in the liver and serum, carry out a large partof the catabolic process. Reduction reactions occur mainlyintracellularly in the endoplasmic reticulum.

Phase II enzymes detoxify toxic substances by catalyzing theirconjugation with water-soluble substances, thus increasing toxins'solubility in water and increasing their rate of excretion.Additionally, conjugation reduces the toxins' biological reactivity.Examples of phase II enzymes include glutathione S-transferases andUDP-glucuronosyl transferases, which catalyze conjugation to glutathioneand glucuronic acid, respectively. Transferases perform conjugationreactions mainly in the kidneys and liver.

The liver is the primary site of elimination of most drugs, includingpsychoactive drugs, and contains a plurality of both phase I and phaseII enzymes that oxidize or conjugate drugs, respectively.

Physicians currently prescribe drugs and their dosages based on apopulation average and fail to take genetic variability into account.The variability between individuals in drug metabolism is usually due toboth genetic and environmental factors, in particular, how thedrug-metabolizing enzymes are controlled. With certain enzymes, thegenetic component predominates and variability is associated withvariants of the normal, wild-type enzyme.

Most drug-metabolizing enzymes exhibit clinically relevant geneticpolymorphisms. Essentially all of the major human enzymes responsiblefor modification of functional groups or conjugation with endogenoussubsituents exhibit common polymorphisms at the genomic level. Forexample, polymorphisms expressing a non-functioning variant enzymeresults in a sub-group of patients in the population who are more proneto the concentration-dependent effects of a drug. This sub-group ofpatients may show toxic side effects to a dose of drug that is otherwisewithout side effects in the general population. Recent development ingenotyping allows identification of affected individuals. As a result,their atypical metabolism and likely response to a drug metabolized bythe affected enzyme can be understood and predicted, thus permitting thephysician to adjust the dose of drug they receive to achieve improvedtherapy.

A similar approach is also becoming important in identifying riskfactors associated with the development of various cancers. This isbecause the enzymes involved in drug metabolism are also responsible forthe activation and detoxification of chemical carcinogens. Specifically,the development of neoplasia is regulated by a balance between phase Ienzymes, which activate carcinogens, and phase II enzymes, whichdetoxify them. Accordingly, an individual's susceptibility to canceroften involves the balance between these two processes, which is, inpart, genetically determined and can be screened by suitable genotypingtests. Higher induction of phase I enzymes compared to phase II enzymesresults in the generation of large amounts of electrophiles and reactiveoxygen species and may cause DNA and membrane damage and other adverseeffects leading to neoplasia. Conversely, higher levels of phase IIenzyme expression can protect cells from various chemical compounds.

Abnormal activity of drug-metabolizing enzymes has been implicated in arange of human diseases, including cancer, Parkinson's disease, myetonicdystrophy, and developmental defects.

Cytochrome p450

An example of a phase I drug-metabolizing enzyme is the cytochrome p450(“CYP”) superfamily, the members of which comprise the majordrug-metabolizing enzymes expressed in the liver. The CYP superfamilycomprises heme proteins which catalyze the oxidation and dehydrogenationof a number of endogenous and exogenous lipophilic compounds. The CYPsuperfamily has immense diversity in its functions, with hundreds ofisoforms in many species catalyzing many types of chemical reactions.The CYP superfamily comprises at least 30 related enzymes, which aredivided into different families according to their amino acid homology.Examples of CYP families include CYP families 1, 2, 3 and 4, whichcomprise endoplasmic reticulum proteins responsible for the metabolismof drugs and other xenobiotics. Approximately 10-15 individual geneproducts within these four families metabolize thousands of structurallydiverse compounds. It is estimated that collectively the enzymes in theCYP superfamily participate in the metabolism of greater than 80% of allavailable drugs used in humans. For example, the CYP 1A subfamilycomprises CYP 1A2, which metabolizes several widely used drugs,including acetaminophen, amitriptyline, caffeine, clozapine,haloperidol, imipramine, olanzapine, ondansetron, phenacetin,propafenone, propranolol, tacrine, theophylline, verapamil. In addition,CYP enzymes play additional roles in the metabolism of some endogenoussubstrates including prostaglandins and steroids.

Some CYP enzymes exist in a polymorphic form, meaning that a smallpercentage of the population possesses mutant genes that alter theactivity of the enzyme, usually by diminishing or abolishing activity.For example, a genetic polymorphism has been well characterized with theCYP 2C19 and CYP 2D6 genes. Substrates of CYP 2C19 include clomipramine,diazepam, imipramine, mephenyloin, moclobemide, omeprazole, phenyloin,propranolol, and tolbutamide. Substrates of CYP 2D6 include alprenolol,amitriptyline, chlorpheniramine, clomipramine, codeine, desipramine,dextromethorphan, encainide, fluoxetine, haloperidol, imipramine,indoramin, metoprolol, nortriptyline, ondansetron, oxycodone,paroxetine, propranolol, and propafenone. Polymorphic variants of thesegenes metabolize these substrates at different rates, which can effect apatient's effective therapeutic dosage.

While the substrate specificity of CYPs must be very broad toaccommodate the metabolism of all of these compounds, each individualCYP gene product has a narrower substrate specificity defined by itsbinding and catalytic sites. Drug metabolism can thereby be regulated bychanges in the amount or activity of specific CYP gene products. Methodsof CYP regulation include genetic differences in the expression of CYPgene products (i.e., genetic polymorphisms), inhibition of CYPmetabolism by other xenobiotics that also bind to the CYP, and inductionof certain CYPs by the drug itself or other xenobiotics. Inhibition andinduction of CYPs is one of the most common mechanisms of adverse druginteractions. For example, the CYP3A subfamily is involved in clinicallysignificant drug interactions involving nonsedating antihistamines andcisapride that may result in cardiac dysrhythmias. In another example,CYP3A4 and CYP1A2 enzymes are involved in drug interactions involvingtheophylline. In yet another example, CYP2D6 is responsible for themetabolism of many psychotherapeutic agents. Additionallly, CYP enzymesmetabolize the protease inhibitors used to treat patients infected withthe human immunodeficiency virus. By understanding the unique functionsand characteristics of these enzymes, physicians may better anticipateand manage drug interactions and may predict or explain an individual'sresponse to a particular therapeutic regimen.

Examples of reactions catalyzed by the CYP superfamily includeperoxidative reactions utilizing peroxides as oxygen donors inhydroxylation reactions, as substrates for reductive beta-scission, andas peroxyhemiacetal intermediates in the cleavage of aldehydes toformate and alkenes. Lipid hydroperoxides undergo reductivebeta-cleavage to give hydrocarbons and aldehydic acids. One of theseproducts, trans-4-hydroxynonenal, inactivates CYP, particularlyalcohol-inducible 2E1, in what may be a negative regulatory process.Although a CYP iron-oxene species is believed to be the oxygen donor inmost hydroxylation reactions, an iron-peroxy species is apparentlyinvolved in the deformylation of many aldehydes with desaturation of theremaining structure, as in aromatization reactions.

Examples of drugs with oxidative metabolism associated with CYP enzymesinclude acetaminophen, alfentanil, alprazolam, alprenolol, amiodarone,amitriptyline, astemizole, buspirone caffeine, carbamazepine,chlorpheniramine, cisapride, clomipramine, clomipramine, clozapine,codeine, colchicine, cortisol, cyclophosphamide, cyclosporine, dapsone,desipramine, dextromethorphan, diazepam, diclofenac, diltiazem,encainide, erythromycin, estradiol, felodipine, fluoxetine, fluvastatin,haloperidol, ibuprofen, imipramine, indinavir, indomethacin, indoramin,irbesartan, lidocaine, losartan, macrolide antibiotics, mephenyloin,methadone, metoprolol, mexilitene, midazolam, moclobemide, naproxen,nefazodone, nicardipine, nifedipine, nitrendipine, nortriptyline,olanzapine, omeprazole, ondansetron, oxycodone, paclitaxel, paroxetine,phenacetin, phenyloin, piroxicam, progesterone, propafenone,propranolol, quinidine, ritonavir, saquinavir, sertraline, sildenafil,S-warfarin, tacrine, tamoxifen, tenoxicam, terfenadine, testosterone,theophylline, timolol, tolbutamide, triazolam, verapamil, andvinblastine.

Abnormal activity of phase I enzymes has been implicated in a range ofhuman diseases. For example, enhanced CYP2D6 activity has been relatedto malignancies of the bladder, liver, pharynx, stomach and lungs,whereas decreased CYP2D activity has been linked to an increased risk ofParkinson's disease. Other syndromes and developmental defectsassociated with deficiencies in the CYP superfamily includecerebrotendinous xanthomatosis, adrenal hyperplasia, gynecomastia, andmyetonic dystrophy.

The CYP superfamily a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of the CYPsuperfamily.

UDP-Glycosyltransferases

Potential drug interactions involving phase II metabolism areincreasingly being recognized. An important group of phase II enzymesinvolved in drug metabolism are the glucuronosyltransferases, especiallythe UDP-glycosyltransferase (alternatively referred to asUDP-glucuronosyltransferase) (“UGT”) superfamily. The novel humanprotein, and encoding gene, provided by the present invention shows thehighest degree of similarity to UGT2B 11. Members of the UGT2B familyare important for conjugating glucuronic acid to bile salts and steroidhormones, as well as to phenol derivatives, bilirubin, and fatty acids.For a further review of UGT2B11, see Beaulieu et al., Biochem BiophysRes Commun 1998 Jul. 9; 248(1): 44-50 and Jin et al., Biochem. Biophys.Res. Commun. 194: 496-503, 1993.

Members of the UGT superfamily catalyze the enzymatic addition of UDPglucuronic acid as a sugar donor to fat-soluble chemicals, a processthat increases their solubility in water and increases their rate ofexcretion. In mammals, glucuronic acid is the main sugar that is used toprevent the accumulation of waste products of metabolism and fat-solublechemicals from the environment to toxic levels in the body. Bothinducers and inhibitors of glucuronosyltransferases are known and havethe potential to affect the plasma concentration and actions ofimportant drugs, including psychotropic drugs.

The UGT superfamily comprises several families of enzymes in severalspecies defined with a nomenclature similar to that used to definemembers of the CYP superfamily. In animals, yeast, plants and bacteriathere are at least 110 distinct known members of the UGT superfamily. Asmany as 33 families have been defined, with three families identified inhumans. Different UGT families are defined as having <45% amino acidsequence homology; within subfamilies there is approximately 60%homology. The members of the UGT superfamily are part of a furthersuperfamily of UDP glycosyltransferases found in animals, plants andbacteria.

The role of phase II enzymes, and of UGT enzymes in particular, is beingincreasingly recognized as important in psychopharmacology. UGT enzymesconjugate many important psychotropic drugs and are an important sourceof variability in drug response and drug interactions. For example, thebenzodiazepines lorazepam, oxazepam, and temazepam undergo phase IIreactions exclusively before being excreted into the urine.

Phase II enzymes metabolize and detoxify hazardous substances, such ascarcinogens. The expression of genes encoding phase II enzymes is knownto be up-regulated by hundreds of agents. For example, oltipraz is knownto up-regulate phase II enzyme expression. Studies have demonstratedprotection from the cancer-causing effects of carcinogens when selectedphase II enzyme inducers are administered prior to the carcinogens. Thepotential use of phase II enzyme inducers in humans for prevention ofcancers related to exposure to carcinogens has prompted studies aimed atunderstanding their molecular effects. Current biochemical and molecularbiological research methodologies can be used to identify andcharacterize selective phase II enzyme inducers and their targets.Identification of genes responding to cancer chemopreventive agents willfacilitate studies of their basic mechanism and provide insights aboutthe relationship between gene regulation, enzyme polymorphism, andcarcinogen detoxification.

Examples of drugs with conjugative metabolism associated with UGTenzymes include amitriptyline, buprenorphine, chlorpromazine, clozapine,codeine, cyproheptadine, dihydrocodeine, doxepin, imipramine,lamotrigine, lorazepam, morphine, nalorphine, naltrexone, temazepam, andvalproate.

Abnormal activity of phase II enzymes has been implicated in a range ofhuman diseases. For example, Gilbert syndrome is an autosomal dominantdisorder caused by mutation in the UGT1 gene, and mutations in theUGT1A1 enzyme have been demonstrated to be responsible forCrigler-Najjar syndrome.

Drug-metabolizing enzymes, particularly members of theUDP-glycosyltransferase drug-metabolizing enzyme subfamily, are a majortarget for drug action and development. Accordingly, it is valuable tothe field of pharmaceutical development to identify and characterizepreviously unknown members of this subfamily of drug-metabolizingproteins. The present invention advances the state of the art byproviding previously unidentified human drug-metabolizing proteins thathave homology to members of the UDP-glycosyltransferasedrug-metabolizing enzyme subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human drug-metabolizing enzyme peptides and proteinsthat are related to the UDP-glycosyltransferase drug-metabolizing enzymesubfamily, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate drug-metabolizing enzyme activity in cells andtissues that express the drug-metabolizing enzyme. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodesthe drug-metabolizing enzyme protein of the present invention. (SEQ IDNO:1) In addition, structure and functional information is provided,such as ATG start, stop and tissue distribution, where available, thatallows one to readily determine specific uses of inventions based onthis molecular sequence. Experimental data as provided in FIG. 1indicates expression in humans in the eye, kidney, hepatocellularcarcinoma, and a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample.

FIG. 2 provides the predicted amino acid sequence of thedrug-metabolizing enzyme of the present invention. (SEQ ID NO:2) Inaddition structure and functional information such as protein family,function, and modification sites is provided where available, allowingone to readily determine specific uses of inventions based on thismolecular sequence.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a drug-metabolizing enzyme protein or part of adrug-metabolizing enzyme protein and are related to theUDP-glycosyltransferase drug-metabolizing enzyme subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman drug-metabolizing enzyme peptides and proteins that are related tothe UDP-glycosyltransferase drug-metabolizing enzyme subfamily, andnucleic acid sequences in the form of cDNA/transcript sequences thatencode these drug-metabolizing enzyme peptides and proteins, tissuedistribution of expression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thedrug-metabolizing enzyme of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known drug-metabolizing enzyme proteins of theUDP-glycosyltransferase drug-metabolizing enzyme subfamily and theexpression pattern observed. Experimental data as provided in FIG. 1indicates expression in humans in the eye, kidney, hepatocellularcarcinoma, and a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. The art has clearly established the commercial importance ofmembers of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known UDP-glycosyltransferase family orsubfamily of drug-metabolizing enzyme proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thedrug-metabolizing enzyme family of proteins and are related to theUDP-glycosyltransferase drug-metabolizing enzyme subfamily (proteinsequences are provided in FIG. 2, transcript/cDNA sequences are providedin FIG. 1). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, will be referred herein as thedrug-metabolizing enzyme peptides of the present invention,drug-metabolizing enzyme peptides, or peptides/proteins of the presentinvention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the drug-metabolizing enzyme peptides disclosed in FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA),as well as all obvious variants of these peptides that are within theart to make and use. Some of these variants are described in detailbelow.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thedrug-metabolizing enzyme peptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

The isolated drug-metabolizing enzyme peptide can be purified from cellsthat naturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. For example, anucleic acid molecule encoding the drug-metabolizing enzyme peptide iscloned into an expression vector, the expression vector introduced intoa host cell and the protein expressed in the host cell. The protein canthen be isolated from the cells by an appropriate purification schemeusing standard protein purification techniques. Many of these techniquesare described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the drug-metabolizing enzyme peptides of the presentinvention are the naturally occurring mature proteins. A briefdescription of how various types of these proteins can be made/isolatedis provided below.

The drug-metabolizing enzyme peptides of the present invention can beattached to heterologous sequences to form chimeric or fusion proteins.Such chimeric and fusion proteins comprise a drug-metabolizing enzymepeptide operatively linked to a heterologous protein having an aminoacid sequence not substantially homologous to the drug-metabolizingenzyme peptide. “Operatively linked” indicates that thedrug-metabolizing enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the drug-metabolizing enzyme peptide.

In some uses, the fusion protein does not affect the activity of thedrug-metabolizing enzyme peptide per se. For example, the fusion proteincan include, but is not limited to, enzymatic fusion proteins, forexample beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant drug-metabolizing enzyme peptide. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A drug-metabolizing enzyme peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the drug-metabolizing enzymepeptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the drug-metabolizing enzyme peptides ofthe present invention. The degree of homology/identity present will bebased primarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap; which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48): 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1): 387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17): 3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thedrug-metabolizing enzyme peptides of the present invention as well asbeing encoded by the same genetic locus as the drug-metabolizing enzymepeptide provided herein.

Allelic variants of a drug-metabolizing enzyme peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of thedrug-metabolizing enzyme peptide as well as being encoded by the samegenetic locus as the drug-metabolizing enzyme peptide provided herein.Genetic locus can readily be determined based on the sequenceinformation provided herein, and using sequence-based mapping methodswell known in the art. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a drug-metabolizing enzymepeptide encoding nucleic acid molecule under stringent conditions asmore fully described below.

Paralogs of a drug-metabolizing enzyme peptide can readily be identifiedas having some degree of significant sequence homology/identity to atleast a portion of the drug-metabolizing enzyme peptide, as beingencoded by a gene from humans, and as having similar activity orfunction. Two proteins will typically be considered paralogs when theamino acid sequences are typically at least about 60% or greater, andmore typically at least about 70% or greater homology through a givenregion or domain. Such paralogs will be encoded by a nucleic acidsequence that will hybridize to a drug-metabolizing enzyme peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a drug-metabolizing enzyme peptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the drug-metabolizing enzymepeptide as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a drug-metabolizing enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the drug-metabolizing enzymepeptides of the present invention can readily be generated usingrecombinant techniques. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of thedrug-metabolizing enzyme peptide. For example, one class ofsubstitutions are conserved amino acid substitution. Such substitutionsare those that substitute a given amino acid in a drug-metabolizingenzyme peptide by another amino acid of like characteristics. Typicallyseen as conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247: 1306-1310 (1990).

Variant drug-metabolizing enzyme peptides can be fully functional or canlack function in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244: 1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as drug-metabolizing enzyme activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224: 899-904 (1992); de Vos et al.Science 255: 306-312 (1992)).

The present invention further provides fragments of thedrug-metabolizing enzyme peptides, in addition to proteins and peptidesthat comprise and consist of such fragments, particularly thosecomprising the residues identified in FIG. 2. The fragments to which theinvention pertains, however, are not to be construed as encompassingfragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a drug-metabolizing enzyme peptide.Such fragments can be chosen based on the ability to retain one or moreof the biological activities of the drug-metabolizing enzyme peptide orcould be chosen for the ability to perform a function, e.g. bind asubstrate or act as an immunogen. Particularly important fragments arebiologically active fragments, peptides that are, for example, about 8or more amino acids in length. Such fragments will typically comprise adomain or motif of the drug-metabolizing enzyme peptide, e.g., activesite, a transmembrane domain or a substrate-binding domain. Further,possible fragments include, but are not limited to, domain or motifcontaining fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in drug-metabolizingenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol 182:626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663: 48-62(1992)).

Accordingly, the drug-metabolizing enzyme peptides of the presentinvention also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code, in which asubstituent group is included, in which the mature drug-metabolizingenzyme peptide is fused with another compound, such as a compound toincrease the half-life of the drug-metabolizing enzyme peptide (forexample, polyethylene glycol), or in which the additional amino acidsare fused to the mature drug-metabolizing enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature drug-metabolizing enzyme peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a drug-metabolizingenzyme-effector protein interaction or drug-metabolizing enzyme-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, drug-metabolizing enzymes isolated from humansand their human/mammalian orthologs serve as targets for identifyingagents for use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the drug-metabolizing enzyme. Experimentaldata as provided in FIG. 1 indicates that drug-metabolizing proteins ofthe present invention are expressed in humans in the eye, kidney, andhepatocellular carcinoma, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. A large percentage of pharmaceutical agents are being developedthat modulate the activity of drug-metabolizing enzyme proteins,particularly members of the UDP-glycosyltransferase subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in humansin the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

The drug-metabolizing enzyme polypeptides (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to drug-metabolizing enzymesthat are related to members of the UDP-glycosyltransferase subfamily.Such assays involve any of the known drug-metabolizing enzyme functionsor activities or properties useful for diagnosis and treatment ofdrug-metabolizing enzyme-related conditions that are specific for thesubfamily of drug-metabolizing enzymes that the one of the presentinvention belongs to, particularly in cells and tissues that express thedrug-metabolizing enzyme. Experimental data as provided in FIG. 1indicates that drug-metabolizing proteins of the present invention areexpressed in humans in the eye, kidney, and hepatocellular carcinoma, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

The drug-metabolizing enzyme polypeptides are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the drug-metabolizingenzyme, as a biopsy or expanded in cell culture. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe drug-metabolizing enzyme protein.

The polypeptides can be used to identify compounds that modulatedrug-metabolizing enzyme activity of the protein in its natural state oran altered form that causes a specific disease or pathology associatedwith the drug-metabolizing enzyme. Both the drug-metabolizing enzymes ofthe present invention and appropriate variants and fragments can be usedin high-throughput screens to assay candidate compounds for the abilityto bind to the drug-metabolizing enzyme. These compounds can be furtherscreened against a functional drug-metabolizing enzyme to determine theeffect of the compound on the drug-metabolizing enzyme activity.Further, these compounds can be tested in animal or invertebrate systemsto determine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) the drug-metabolizingenzyme to a desired degree.

Further, the drug-metabolizing enzyme polypeptides can be used to screena compound for the ability to stimulate or inhibit interaction betweenthe drug-metabolizing enzyme protein and a molecule that normallyinteracts with the drug-metabolizing enzyme protein. Such assaystypically include the steps of combining the drug-metabolizing enzymeprotein with a candidate compound under conditions that allow thedrug-metabolizing enzyme protein, or fragment, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the drug-metabolizing enzyme protein and the target.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354: 82-84 (1991);Houghten et al., Nature 354: 84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72: 767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantdrug-metabolizing enzymes or appropriate fragments containing mutationsthat affect drug-metabolizing enzyme function and thus compete forsubstrate. Accordingly, a fragment that competes for substrate, forexample with a higher affinity, or a fragment that binds substrate butdoes not allow release, is encompassed by the invention.

Any of the biological or biochemical functions mediated by thedrug-metabolizing enzyme can be used as an endpoint assay. These includeall of the biochemical or biochemical/biological events describedherein, in the references cited herein, incorporated by reference forthese endpoint assay targets, and other functions known to those ofordinary skill in the art or that can be readily identified using theinformation provided in the Figures, particularly FIG. 2. Specifically,a biological function of a cell or tissues that expresses thedrug-metabolizing enzyme can be assayed. Experimental data as providedin FIG. 1 indicates that drug-metabolizing proteins of the presentinvention are expressed in humans in the eye, kidney, and hepatocellularcarcinoma, as indicated by virtual northern blot analysis. In addition,PCR-based tissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

Binding and/or activating compounds can also be screened by usingchimeric drug-metabolizing enzyme proteins in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native drug-metabolizing enzyme. Accordingly, adifferent set of signal transduction components is available as anend-point assay for activation. This allows for assays to be performedin other than the specific host cell from which the drug-metabolizingenzyme is derived.

The drug-metabolizing enzyme polypeptides are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the drug-metabolizing enzyme (e.g. binding partners and/orligands). Thus, a compound is exposed to a drug-metabolizing enzymepolypeptide under conditions that allow the compound to bind or tootherwise interact with the polypeptide. Soluble drug-metabolizingenzyme polypeptide is also added to the mixture. If the test compoundinteracts with the soluble drug-metabolizing enzyme polypeptide, itdecreases the amount of complex formed or activity from thedrug-metabolizing enzyme target. This type of assay is particularlyuseful in cases in which compounds are sought that interact withspecific regions of the drug-metabolizing enzyme. Thus, the solublepolypeptide that competes with the target drug-metabolizing enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the drug-metabolizing enzyme protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofdrug-metabolizing enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a drug-metabolizing enzyme-binding protein and a candidate compoundare incubated in the drug-metabolizing enzyme protein-presenting wellsand the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the drug-metabolizing enzymeprotein target molecule, or which are reactive with drug-metabolizingenzyme protein and compete with the target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the target molecule.

Agents that modulate one of the drug-metabolizing enzymes of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

Modulators of drug-metabolizing enzyme protein activity identifiedaccording to these drug screening assays can be used to treat a subjectwith a disorder mediated by the drug-metabolizing enzyme pathway, bytreating cells or tissues that express the drug-metabolizing enzyme.Experimental data as provided in FIG. 1 indicates expression in humansin the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. These methods oftreatment include the steps of administering a modulator ofdrug-metabolizing enzyme activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

In yet another aspect of the invention, the drug-metabolizing enzymeproteins can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi etal. (1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identifyother proteins, which bind to or interact with the drug-metabolizingenzyme and are involved in drug-metabolizing enzyme activity. Suchdrug-metabolizing enzyme-binding proteins are likely to bedrug-metabolizing enzyme inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for adrug-metabolizing enzyme protein is fused to a gene encoding the DNAbinding domain of a known transcription factor (e.g., GAL-4). In theother construct, a DNA sequence, from a library of DNA sequences, thatencodes an unidentified protein (“prey” or “sample”) is fused to a genethat codes for the activation domain of the known transcription factor.If the “bait” and the “prey” proteins are able to interact, in vivo,forming a drug-metabolizing enzyme-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the drug-metabolizingenzyme protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a drug-metabolizing enzyme-modulating agent, anantisense drug-metabolizing enzyme nucleic acid molecule, adrug-metabolizing enzyme-specific antibody, or a drug-metabolizingenzyme-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The drug-metabolizing enzyme proteins of the present invention are alsouseful to provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the drug-metabolizing enzyme protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered drug-metabolizing enzymeactivity in cell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11): 983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2): 254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the drug-metabolizing enzyme proteinin which one or more of the drug-metabolizing enzyme functions in onepopulation is different from those in another population. The peptidesthus allow a target to ascertain a genetic predisposition that canaffect treatment modality. Thus, in a ligand-based treatment,polymorphism may give rise to amino terminal extracellular domainsand/or other substrate-binding regions that are more or less active insubstrate binding, and drug-metabolizing enzyme activation. Accordingly,substrate dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Accordingly,methods for treatment include the use of the drug-metabolizing enzymeprotein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe drug-metabolizing enzyme proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/ordrug-metabolizing enzyme/binding partner interaction. FIG. 2 can be usedto identify particularly important regions while sequence alignment canbe used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatdrug-metabolizing proteins of the present invention are expressed inhumans in the eye, kidney, and hepatocellular carcinoma, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the eye, kidney, hepatocellular carcinoma, and amixed brain/heart/kidney/lung/spleen/testis/leukocyte sample. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The diagnosticuses can be applied, not only in genetic testing, but also in monitoringa treatment modality. Accordingly, where treatment is ultimately aimedat correcting expression level or the presence of aberrant sequence andaberrant tissue distribution or developmental expression, antibodiesdirected against the protein or relevant fragments can be used tomonitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the drug-metabolizing enzyme peptide toa binding partner such as a substrate. These uses can also be applied ina therapeutic context in which treatment involves inhibiting theprotein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a drug-metabolizing enzyme peptide or protein of the presentinvention (cDNA/transcript sequence provided in FIG. 1). Such nucleicacid molecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the drug-metabolizing enzymepeptides of the present invention, an allelic variant thereof, or anortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5 KB, 4 KB,3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1,cDNA/transcript sequence), or any nucleic acid molecule that encodes theprotein provided in FIG. 2, SEQ ID NO:2. A nucleic acid moleculeconsists of a nucleotide sequence when the nucleotide sequence is thecomplete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1, cDNA/transcript sequence), or any nucleic acid molecule thatencodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acidmolecule consists essentially of a nucleotide sequence when such anucleotide sequence is present with only a few additional nucleic acidresidues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 (SEQ ID NO:1,cDNA/transcript sequence), or any nucleic acid molecule that encodes theprotein provided in FIG. 2, SEQ ID NO:2. A nucleic acid moleculecomprises a nucleotide sequence when the nucleotide sequence is at leastpart of the final nucleotide sequence of the nucleic acid molecule. Insuch a fashion, the nucleic acid molecule can be only the nucleotidesequence or have additional nucleic acid residues, such as nucleic acidresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have a fewadditional nucleotides or can comprises several hundred or moreadditional nucleotides. A brief description of how various types ofthese nucleic acid molecules can be readily made/isolated is providedbelow.

In FIG. 1, both coding and non-coding sequences are provided. Thenucleic acid molecules in the Figures may contain 5′ and 3′ non-codingsequences, unspliced intronic sequences, and gene regulatory regions. Ingeneral such sequence features are either noted in FIG. 1 or can readilybe identified using computational tools known in the art. As discussedbelow, some of the non-coding regions, particularly gene regulatoryelements such as promoters, are useful for a variety of purposes, e.g.control of heterologous gene expression, target for identifying geneactivity modulating compounds, and are particularly claimed as fragmentsof the nucleic acid sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the drug-metabolizing enzymepeptide alone, the sequence encoding the mature peptide and additionalcoding sequences, such as a leader or secretory sequence (e.g., apre-pro or pro-protein sequence), the sequence encoding the maturepeptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the drug-metabolizingenzyme proteins of the present invention that are described above. Suchnucleic acid molecules may be naturally occurring, such as allelicvariants (same locus), paralogs (different locus), and orthologs(different organism), or may be constructed by recombinant DNA methodsor by chemical synthesis. Such non-naturally occurring variants may bemade by mutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIG. 1. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thecDNA/transcript sequence provided in FIG. 1.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatdrug-metabolizing proteins of the present invention are expressed inhumans in the eye, kidney, and hepatocellular carcinoma, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Accordingly, theprobes can be used to detect the presence of, or to determine levels of,a specific nucleic acid molecule in cells, tissues, and in organisms.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in drug-metabolizing enzyme proteinexpression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a drug-metabolizing enzyme protein, suchas by measuring a level of a drug-metabolizing enzyme-encoding nucleicacid in a sample of cells from a subject e.g., mRNA or genomic DNA, ordetermining if a drug-metabolizing enzyme gene has been mutated.Experimental data as provided in FIG. 1 indicates that drug-metabolizingproteins of the present invention are expressed in humans in the eye,kidney, and hepatocellular carcinoma, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate drug-metabolizing enzyme nucleic acidexpression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe drug-metabolizing enzyme gene, particularly biological andpathological processes that are mediated by the drug-metabolizing enzymein cells and tissues that express it. Experimental data as provided inFIG. 1 indicates expression in humans in the eye, kidney, hepatocellularcarcinoma, and a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. The method typically includes assaying the ability of thecompound to modulate the expression of the drug-metabolizing enzymenucleic acid and thus identifying a compound that can be used to treat adisorder characterized by undesired drug-metabolizing enzyme nucleicacid expression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing thedrug-metabolizing enzyme nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

Thus, modulators of drug-metabolizing enzyme gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof drug-metabolizing enzyme mRNA in the presence of the candidatecompound is compared to the level of expression of drug-metabolizingenzyme mRNA in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate drug-metabolizing enzyme nucleic acidexpression in cells and tissues that express the drug-metabolizingenzyme. Experimental data as provided in FIG. 1 indicates thatdrug-metabolizing proteins of the present invention are expressed inhumans in the eye, kidney, and hepatocellular carcinoma, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

Alternatively, a modulator for drug-metabolizing enzyme nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the drug-metabolizing enzyme nucleic acid expression in thecells and tissues that express the protein. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe drug-metabolizing enzyme gene in clinical trials or in a treatmentregimen. Thus, the gene expression pattern can serve as a barometer forthe continuing effectiveness of treatment with the compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in drug-metabolizing enzyme nucleic acid expression,and particularly in qualitative changes that lead to pathology. Thenucleic acid molecules can be used to detect mutations indrug-metabolizing enzyme genes and gene expression products such asmRNA. The nucleic acid molecules can be used as hybridization probes todetect naturally occurring genetic mutations in the drug-metabolizingenzyme gene and thereby to determine whether a subject with the mutationis at risk for a disorder caused by the mutation. Mutations includedeletion, addition, or substitution of one or more nucleotides in thegene, chromosomal rearrangement, such as inversion or transposition,modification of genomic DNA, such as aberrant methylation patterns orchanges in gene copy number, such as amplification. Detection of amutated form of the drug-metabolizing enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a drug-metabolizing enzymeprotein.

Individuals carrying mutations in the drug-metabolizing enzyme gene canbe detected at the nucleic acid level by a variety of techniques.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91: 360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23: 675-682(1995)). This method can include the steps of collecting a sample ofcells from a patient, isolating nucleic acid (e.g., genomic, mRNA orboth) from the cells of the sample, contacting the nucleic acid samplewith one or more primers which specifically hybridize to a gene underconditions such that hybridization and amplification of the gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. Deletions andinsertions can be detected by a change in size of the amplified productcompared to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a drug-metabolizing enzyme gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantdrug-metabolizing enzyme gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19: 448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36: 127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38: 147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230: 1242 (1985));Cotton et al., PNAS 85: 4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86: 2766 (1989); Cotton etal., Mutat. Res. 285: 125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9: 73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313: 495 (1985)). Examples of other techniques for detectingpoint mutations include selective oligonucleotide hybridization,selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the drug-metabolizingenzyme gene in an individual in order to select an appropriate compoundor dosage regimen for treatment.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol drug-metabolizing enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of drug-metabolizingenzyme protein. An antisense RNA or DNA nucleic acid molecule wouldhybridize to the mRNA and thus block translation of mRNA intodrug-metabolizing enzyme protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of drug-metabolizing enzyme nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired drug-metabolizing enzyme nucleic acid expression.This technique involves cleavage by means of ribozymes containingnucleotide sequences complementary to one or more regions in the mRNAthat attenuate the ability of the mRNA to be translated. Possibleregions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thedrug-metabolizing enzyme protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in drug-metabolizing enzymegene expression. Thus, recombinant cells, which include the patient'scells that have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desireddrug-metabolizing enzyme protein to treat the individual.

The invention also encompasses kits for detecting the presence of adrug-metabolizing enzyme nucleic acid in a biological sample.Experimental data as provided in FIG. 1 indicates that drug-metabolizingproteins of the present invention are expressed in humans in the eye,kidney, and hepatocellular carcinoma, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. For example, the kit can comprise reagents such as a labeled orlabelable nucleic acid or agent capable of detecting drug-metabolizingenzyme nucleic acid in a biological sample; means for determining theamount of drug-metabolizing enzyme nucleic acid in the sample; and meansfor comparing the amount of drug-metabolizing enzyme nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect drug-metabolizing enzyme protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIG. 1 (SEQ ID NO:1).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the drug-metabolizing enzyme proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe drug-metabolizing enzyme gene of the present invention.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified drug-metabolizing enzyme gene of the presentinvention can be routinely identified using the sequence informationdisclosed herein can be readily incorporated into one of the establishedkit formats which are well known in the art, particularly expressionarrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67: 31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69: 301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185: 60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30: 933-943 (1982)), pJRY88(Schultz et al., Gene 54: 113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBOJ. 6: 187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, appropriate secretion signalsare incorporated into the vector. The signal sequence can be endogenousto the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, the protein can beisolated from the host cell by standard disruption procedures, includingfreeze thaw, sonication, mechanical disruption, use of lysing agents andthe like. The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing adrug-metabolizing enzyme protein or peptide that can be further purifiedto produce desired amounts of drug-metabolizing enzyme protein orfragments. Thus, host cells containing expression vectors are useful forpeptide production.

Host cells are also useful for conducting cell-based assays involvingthe drug-metabolizing enzyme protein or drug-metabolizing enzyme proteinfragments, such as those described above as well as other formats knownin the art. Thus, a recombinant host cell expressing a nativedrug-metabolizing enzyme protein is useful for assaying compounds thatstimulate or inhibit drug-metabolizing enzyme protein function.

Host cells are also useful for identifying drug-metabolizing enzymeprotein mutants in which these functions are affected. If the mutantsnaturally occur and give rise to a pathology, host cells containing themutations are useful to assay compounds that have a desired effect onthe mutant drug-metabolizing enzyme protein (for example, stimulating orinhibiting function) which may not be indicated by their effect on thenative drug-metabolizing enzyme protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of adrug-metabolizing enzyme protein and identifying and evaluatingmodulators of drug-metabolizing enzyme protein activity. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the drug-metabolizing enzymeprotein nucleotide sequences can be introduced as a transgene into thegenome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the drug-metabolizing enzyme proteinto particular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89: 6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251: 1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385: 810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring born of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, drug-metabolizing enzyme protein activation, and signaltransduction, may not be evident from in vitro cell-free or cell-basedassays. Accordingly, it is useful to provide non-human transgenicanimals to assay in vivo drug-metabolizing enzyme protein function,including substrate interaction, the effect of specific mutantdrug-metabolizing enzyme proteins on drug-metabolizing enzyme proteinfunction and substrate interaction, and the effect of chimericdrug-metabolizing enzyme proteins. It is also possible to assess theeffect of null mutations, that is mutations that substantially orcompletely eliminate one or more drug-metabolizing enzyme proteinfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

1. An isolated peptide consisting of an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence shown in SEQ IDNO:2; (b) an amino acid sequence of an allelic variant of an amino acidsequence shown in SEQ ID NO:2, wherein said allelic variant is encodedby a nucleic acid molecule that hybridizes under stringent conditions tothe opposite strand of a nucleic acid molecule shown in SEQ ID NO:1; (c)an amino acid sequence of an ortholog of an amino acid sequence shown inSEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NO:1; and (d) a fragment of anamino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids.
 2. An isolated peptidecomprising an amino acid sequence selected from the group consisting of:(a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acidsequence of an allelic variant of an amino acid sequence shown in SEQ IDNO:2, wherein said allelic variant is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NO:1; (c) an amino acid sequenceof an ortholog of an amino acid sequence shown in SEQ ID NO:2, whereinsaid ortholog is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NO:1; and (d) a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NO:1; (c) anucleotide sequence that encodes an ortholog of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NO:1; (d) a nucleotide sequence that encodes a fragmentof an amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NO:1; (c) a nucleotide sequence that encodes an orthologof an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NO:1; (d) a nucleotide sequencethat encodes a fragment of an amino acid sequence shown in SEQ ID NO:2,wherein said fragment comprises at least 10 contiguous amino acids; and(e) a nucleotide sequence that is the complement of a nucleotidesequence of (a)-(d).
 6. A gene chip comprising a nucleic acid moleculeof claim
 5. 7. A transgenic non-human animal comprising a nucleic acidmolecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acidmolecule of claim
 5. 9. A host cell containing the vector of claim 8.10. A method for producing any of the peptides of claim 1 comprisingintroducing a nucleotide sequence encoding any of the amino acidsequences in (a)-(d) into a host cell, and culturing the host cell underconditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human drug-metabolizing enzymeprotein, said method comprising administering to a patient apharmaceutically effective amount of an agent identified by the methodof claim
 16. 19. A method for identifying a modulator of the expressionof a peptide of claim 2, said method comprising contacting a cellexpressing said peptide with an agent, and determining if said agent hasmodulated the expression of said peptide.
 20. An isolated humandrug-metabolizing enzyme peptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human drug-metabolizingenzyme peptide, said nucleic acid molecule sharing at least 80 percenthomology with a nucleic acid molecule shown in SEQ ID NO:1.
 23. Anucleic acid molecule according to claim 22 that shares at least 90percent homology with a nucleic acid molecule shown in SEQ ID NO:1.